Research and Reviews ill Parasitology. 60 (3-4): 113-119 (2000) Published by A.P.E. © 2000 Asociaci6n de Parasit61ogos Espafioles (A.P.E.) Printed in Barcelona.

EPIDEMIOLOGY OF LEISHMANIOSIS IN THE NATURE RESERVE OFSIERRA DEARACENA AND PICOS DE (SOUTHWEST SPAIN)

i 2 3 3 i J.A. , J.M. UBEDA , F. MORILLAS , D.e. GUEVARAZ, J. MARTIN-SANCHEZ , F.J. GUERRERO , M.C. SANCHIS-MARI~ & R. PEREAI

J Hospital General de Riotinto, Riotinto, , Spain 20epartamento de Microbiologia y Parasitologia, Facultad de Farmacia, Universidad de Sevilla, Cl Pro! Garcia Gonzale: sin .. 41012 Sevilla, Spain 30eparramento de Parasitologia, Facultad de Farmacia, Universidad de Granada, Campus La Carruja, 18011 Granada, Spain "Departamento de Biologia Animal, Facultad de Ciencias Experimentales, Universidad de Almeria, Almeria, Spain

Received 25 May 1999; accepted 17 November 1999

REFERENCE:LEPE(J.A.), UBEDA(J.M.), MORILLAS(F.). GUEVARA(D.C.), MARllN-SANCHEJZ( .), GUERRERO.(FJ.), SANCI-Us-MARIM.CN( .) & PE· REA(R.), 2000.- Epidemiology of leishmaniosis in the Nature Reserve of Sierra de and Picos de Aroche (Southwest Spain). Research and Reviews in Parasitology, 60 (3-4): 113-119. ABSTRACT:An eco-epiderniological study of leishmaniosis was carried out in the Nature Reserve of Sierra de Aracena and Picos de Aroche, situa- ted in the north of the (southwest Spain). This survey includes a study of the vector, the canine reservoir and the rate of Leish- mania-human contact in the area. Of the sandtly species of proven vectorial capacity, the most abundant was Phlebotomus perniciosus (90,36%) fo- llowed by Phlebotomus ariasi (9,43%). The populations of both species develop over the year according to a typical diphasic curve. A serologic study using lndirect Fluorescent Antibody Test on 702 dogs revealed 47 with a titer ~160 (seroprevalence of 6,7%). Performing a parasitological study on several dogs with a titer ~80 and using electrophoresis analysis of isoenzymes, Leishmania infantum was isolated and identified as respon- sible for the disease. To determine the rate of human contact with Leishmania, the Montenegro skin test was used. Of 413 people tested, 12 were positive (2,9%). This result is not in agreement with the close correlation between canine leishmaniosis and positive Montenegro skin test reported by other authors in the south of Spain. This is probably due to the special relationship between the canine reservoir and human host found in this na- ture reserve, where packs of dogs are kept in kennels well away from houses, and dogs are put down at the first sign of disease, even when unspeci- fic. Furthermore, there are never, or very rarely, houses with stables for working animals, thus avoiding breeding grounds for the vector near hu- mans. KEYWORDS:Seroprevalence, canine leishmaniosis, leishmanin skin test, sandtlies, Southwest Spain.

INTRODUCTION be a potential risk zone for the development of leishma- niosis (MARTINEZ-ORTEGA & CONESA, 1989). Leishmaniosis is a disease caused by different species of flagellates of the Leishmania Ross, 1903 genus. This disease has different clinical aspects and its epidemiologi- MATERIAL ANDMETHODS cal patterns are diverse. The World Health Organization considers leishmaniosis as the tenth parasitic infection in Area of study: The Nature Reserve of Sierra de Aracena and Picos the world. Its morbidity was estimated at 1.200.000 cases de Aroche is situated in the southwest of Spain, in the western part of , and occupies all the north of the province of Huelva per year and its mortality at about one per 1000 cases (Fig. I). It has an area of 186000 hectares and has a population of (WHO, 1984). These data, however, should be reasses- 44334, the density being 15 inhabitants/km" The ratio between sed, since the outbreak of AIDS has brought about a con- men and women is about 1:I. siderable increase in mortality, owing to the fact that 2% From a bioclirnatic point of view, the Nature Reserve is found in of AIDS patients present clinical leishmaniosis (WHO, a IV(V) bioclimatic zone (RJvAs-MARTl EZ, 1988), which corres- 1991). More recent studies emphasize the importance of ponds to a sub-humid Mediterranean climate with an Atlantic in- the coinfection Leishmania-HIV, as it is frequent to find fluence. The mean temperatures range between 13° and 15° C. There is a dry season from June to the end of September and a wet 10% of parasitism in HIV positive patients in Spain (ME- season from October to March, with the heaviest rainfall in De- DRANO et al., 1992). cember and January. This paper is an attempt to get to know the epidemio- logy of leishmaniosis in an area unstudied until now. Study of the vector: Sandfly specimens were captured with sticky This nature reserve is interesting for the study of this di- tape traps during one year in ten stations spread over the Nature sease because there are a great many important packs of Reserve in order to determine the existing species, their density (number of specimens/m? of trap), abundance (number of speci- hunting dogs, and this nature reserve lies in a type IV mens of a species in relation to the total), frequency (number of bioclimatic zone (meso-Mediterranean areas with vege- stations in which a given species has been captured in relation to tation comprising forests of cork-oaks, holm-oaks and the total number of stations) and phenology. Furthermore, a survey leshew Pistacia lentiscus), all of which are considered to was carried out using (eDC) light traps through the years 1995, 114 J.A. LEPE et al.

......

...... : " . .' SPAIN

NATURE RESERVE

2•

Fig. 1.- The Nature Reserve of Sierra de Aracena and Picos de Aroche. I = Aracena; 2 = Jabuguillo; 3 = .

1996 and 1997 in order to determine the percentage of sandflies (1992) and the formula developed by DYE (1992) and DYE et al. parasitised by Leishmania sp. (1993) for a homogeneous canine population (all dogs in similar The captured sandflies were classified according to «ad hoc» epidemiological conditions). This model requires a focus where keys (GIL COLLADO,MORILLAS-MARQUEZ& SANCHIS-MARIN, the percentage of infected sandflies is insignificant. In these condi- 1989; GALLEGO-BERENGUEetR al., 1992) and were dissected follo- tions Ro = SO", and SO is the rate of susceptible dogs (negative). wing the Rroux et at. (1986) technique. Parasitological study of dogs: ine dogs with positive serology Serological study of dogs: A randomized study of 702 dogs be- to L. infantum and with titers ranging from 80 to 10240 were selec- longing to packs of hunting hounds was performed. Samples were ted for parasitological study. Nine popliteus ganglion biopsies and obtained from cephalic vein and tested by IFAT. An antigen of the two skin biopsies were made. The puncture of the popliteus gan- Leishmania infantum Nicolle, 1909 strain MCANlES/9OIDP204 glion was performed with needle and syringe after disinfecting the was used at a concentration of 2 X 106 promastigotes/ml. Titres of skin. The skin biopsies were made on the dermatitis areas with the 20,40, 80, and 160 were taken into account. Sera with a titer 20 help of a 4 mm biopsy punch after disinfecting the area. In both ca- were considered seropositive and only titres ~16O were used to de- ses the samples were treated with a solution of sodic Penicillin G termine the seroprevalence. and later inoculated in a NNN medium, which was supplemented The Basic Reproduction Number (Ro) was determined follo- with fetal bovine serum and liquid Evans Modified Tobie's Me- wing the mathematical model of HASIBEDER,DYE & CARPENTIER dium (EMTM). Leishmaniosis in the province of Huelva (Spain) 115

Species Male Female Total Abundance Frequency

Phlebotomus perniciosus 1925 184 2109 48,9% 10110 Sergentomyia minuta 969 1013 1982 45,9% lOll 0 Phlebotomus ariasi 209 11 220 5,1% 8/10 Phlebotomus sergenti 2 I 3 0,06% 3/10 Phlebotomus papatasi 2 0 2 0,04% 1/10

Total 3107 1209 4316

Table 1.- Sandflies captured.

Study of the human population using the Montenegro skin P. perniciosus P. ariasi test: In order to determine the incidence of contact between L. in- Date Specimens/m! Specimens/m' fantum and the human population in the Nature Reserve, a study of cellular immunity was carried out using the Montenegro skin test April, 14th 0,1 (leishmanin skin test) (WHO, 1984). Authorised by the Delega- April, 25th ci6n Provincial del Servicio Andaluz de Salud, a survey was ca- May, 15th 0,7 0,2 rried out on 413 people (256 children and 157 adults), which is May, 31th 0,2 about 1% of the population. All children required written consent June, 14th 9,9 0,2 from their parents to be enrolled. June, 30th 28,9 1,1 The skin test was made using a Dermojet® and 0,1 ml of leish- July, 12th 27,3 2,0 manin was inoculated in the upper arm. The test was examined at July, 30th 9,1 0,6 48 hours. Indurations of mm or more were considered positive 5 Aug., 15th 9,5 0,3 (ACEDO-SANCHEZ et al., 1996). Aug., 30th 12,2 1,5 Sept., 13th 13,9 0,3 Retrospective study of leishmaniosis cases: In order to know the Sept.,30th 42,7 1,3 incidence of the disease in the area, all the clinical histories of the Oct., 15th 28,0 5,8 last ten years (1987-1997) from the Hospital General de Riotinto, o«., 30th 10,1 3,1 as well as clinical histories of the patients from the Dermatology Nov., 14th 0,6 1,0 Department of our reference hospital in the city of Huelva, were Nov., 30th 0,2 reviewed. Dec., 14th to March 30th

Statistical study: In this study l and the exact Fisher test were used. The results were stored and processed in an EPIINFO 6.04 Table 2.-Density of Phlebotomus perniciosus and Phlebotomus epidemiological program. ariasi.

RESULTS biopsied dogs (nine from the popliteal ganglion and two from the skin) revealed the presence of this protozoa in dogs. Electrophoresis of isoenzymes showed that it was Study of the vector L. infantum. In Table 1 the captured species are seen along with their abundance and frequency. Density of Phlebotomus Study on humans perniciosus Newstead, 1911 and Phlebotomus ariasi Tonnoir, 1921 throughout the period of sampling is ex- As can be seen in Table 4, the mean percentage of po- pressed in Table 2. sitivity to leishmanin was 2,9% (95% confidence inter- val, 1,2 to 4,5) and ranged from 0,8%(95% confidence interval, 0,0 to 1,9) observed in children, and 6,3% in Serological study of dogs adults (95% confidence interval, 2,5 to 11,1). No signi- Out of a total of 702 dogs tested, 308 (43,9%) showed ficant differences were found between men and women, a positive titer against L. infantum, and of these, 47 but there were between children and adults (p = (6,73%) had titers greater or equal to 160. The Ro was 0,0015). 1,78 for titer ~20, and 1,07 for titer ~160. In Table 3 the distribution of serological titers and the value of Ro for Retrospective study of human cases each of them can be seen. From the review of the clinical histories of patients at the hospital, one case of visceral leishmaniosis was Parasitological study found in an HIV positive patient and three cases of cuta- Isolations of Leishmania from all the samples from neous leishmaniosis. 116 I.A. LE PE et al.

Titer 0 20 40 80 ~ 160 activity was found from the middle of May to the middle umber 394 110 80 71 47 of November, a period which coincides with the findings (%) (56,1%)(15,7%) (11,4%) (10.1%)(6,7%) of all the other authors, with the exception of MOR1LLAS Ro 1,78 1,39 1,20 1,07 MARQUEZet al. (1983), who captured this species all year round in subtropical climate stations on the coast of Table 3.- Titers obtained and Ro in dogs. the province of Granada. In the study of the annual fluctuation of P. pernicio- sus populations (Fig. 2), our results are in agreement with those obtained by other authors (MORILLAS-MAR- n Positive % P QUEZet al., 1983; SANCHIS-MARIN,VrLLEGAS& MORI- Children 256 2 0,78 P = 0,0015 LLAS-MARQUEZ,1986; MARTINEZ-ORTEGAet aI., 1982; Adults 157 10 6,36 Significant* CONESA-GALLEGO, ROMERA-LoZANO & MARTINEZ- ORTEGA, 1995) who also obtained a typical diphasic Total 413 12 2,90 curve with two peaks of maximum density at the begin- ning and the end of the summer. The same can be said Table 4.- Montenegro skin test scores. * p < 0,05. Exact Fisher about the type of curve obtained for P. ariasi (Fig. 3), test. although the peaks of maximum density are less accen- tuated and appear in the middle of June and at the end of October. DISCUSSION In order to determine the vectorial role of the different species of sandflies in the Nature Reserve, 244 speci- mens were captured using light traps, of which 95,9% Study of the vector were P. perniciosus, 3,7% were P. ariasi and 0,4% were Among the species of proven capacity to transmit P. sergenti. This distribution of the captures agrees with Leishmania, the most captured in the Nature Reserve that found by other research workers who used the same was P. perniciosus (2109 specimens = 90,36%) follo- method (LUCIENTES-CURDIet aI., 1988; MARTINEZ-OR- wed by P. ariasi (220 specimens = 9,43%). The Phlebo- TEGAet aI., 1991; SANCHIS-MARINet aI., 1997). Howe- tomus sergenti Parrot, 1917 and Phlebotomus papatasi (Scopoli, 1786) captured were insignificant (only three and two specimens, respectively). On the other hand, it 60 is important to point out that 62% of the P. perniciosus specimens were captured in the sampling stations of Hi- 50 guera de la Sierra and Aracena, near the townships of Higuera de la Sierra, Aracena and Jabuguillo. As to the order in which the species are distributed in relation to their abundance, our study does not coincide with any of the previous studies performed in the Iberian 10 Peninsula. In contrast to our findings with P. perniciosus as the most abundant and frequent species, other authors, O~~dL~-.--.--.--.--.~.-~~,-~-+o using the same method of capture, have always found 't-4:'" ~"' .•• \v<>" \~ .•• 't-v'lO s"i$:· cf". ~o~· <:f". \",<>. ",6'. ~",<.i5> Sergentomyia minuta (Rondani, 1843) to occupy the first place. This result is difficult to interpret, since the abun- Fig. 2.- Annual fluctuation of Phlebotomus perniciosus popula- dance of a species can be determined by multiple factors tions. and here these factors have not been taken into account. On the other hand, taking into consideration the featu- res of the Nature Reserve (humid zone with Atlantic in- 10 fluence) attention is drawn to the scarcity of captures of 9 P. ariasi (5,1 %), a species especially abundant in areas 8 7 of these climatic characteristics (Hourn, 1965). Regarding the period of activity of P. perniciosus, this 1 6 .5 5 species was captured in the Nature Reserve from the 8. 4 middle of April to the middle of November. These re- were carried out, or to the peculiarities of the biotopes where the captures were made. Regarding P. ariasi, the Fig. 3.- Annual fluctuation of Phlebotomus ariasi populations. Leishmaniosis in the province of Huelva (Spain) 117 ver, SEMIAO SANTOS et al. (1995) reported in Evora obtained in our work, which reveal that 82,9% of dogs (Portugal), at only a hundred kilometers from our study with titers ~160 are found in the foci of Higuera de la area, that the highest percentages of captures correspond Sierra, Aracena and Jabuguillo, which are included wit- to P. sergenti (42,1 %) and P. ariasi (36,5%) while P. hin the area of influence of the sampling stations «Hi- perniciosus only represented 12% of the total. guera de la Sierra» and «Aracena», where 62% of the Regarding the proportion of infected sandflies, several total number of P. perniciosus were captured. These authors have found up to 5% in their surveys WIRES & observations, together with the fact that among the pos- RIBEIRO,1991; SCHREY,PIRES& MACVEAN, 1989; Mo sible vectors of leishmaniosis, P. perniciosus compri- RILLAS-MARQUEZet al., 1992, 1996). On the other hand, sed 97% of the captures in these stations, would lead us other authors have not detected any sandflies with Leish- to consider this species as the real vector of leishma- mania sp. among the captured specimens (SANCHIS-MA- niosis in the area. RIN et al., 1995; SEMIAO SANTOS et al., 1995). In the present work, Leishmania sp. has not been isolated Parasitological study among the 244 specimens captured and dissected for this purpose. The results of direct diagnosis confirm the indirect se- rological data, proving the presence of L. infantum in the area. Serological study of dogs Studies carried out on the seroprevalence of canine Study of the human population leishmaniosis in the Iberian Peninsula show diverse re- sults, ranging from 0,7% (SEMIAOSANTOSet al., 1995) As can be seen in Table 5, the studies carried out on in Evora (Portugal) to 34% found in the Axarquia region Leishmania-human contact in Spain are few (ACEDO- (Malaga, Spain)(MORILLAS-MARQUEZet al., 1996). SANCHEZet al., 1996; SANCHIS-MARINet al., 1997; Mo The reason for these differences is not only in the diffe- RILLAS-MARQUEZet al., 1996; ALVAR et al., 1996). If rent epidemiological conditions peculiar to each case, we consider these results on the whole and at the same but also in the method of diagnosis and the cut-off titer time from an exclusively geoclimatic point of view, they used by each author as indicative of disease (RSA et al., appear somewhat contradictory. However, when the hu- 1997; MARTINEZCRUZ et al., 1990). man host is included in the medium and when we With the methodology used in the present study, all analyse the changes that it produces in the circumstances the authors are in agreement that a titer ~160 must be which determine the epidemiology of leishmaniosis in considered positive. Therefore, as can be deduced from each zone, these apparently contradictory results can be Table 3, the seroprevalence detected in the dogs stu- explained. Therefore, MORILLAS-MARQUEZet al. (1996) died in the Nature Reserve would be 6,7%. If this result explain their results based on the type of rural buildings is compared with that obtained by other authors in sur- with stables attached to living quarters and consequent veys carried out in the south of the Peninsula, using the abundant breeding ground for the vector in the proximity same methodological criteria, it can be observed that it of man.In the same way, SANCHIS-MARINet al. (1997), is very similar to the 5,3% reported by ACEDO SAN- consider that the low incidence of contact observed in CHEZ et al. (1996) in Alpujarra region (province of their study is due to the wide use of insecticides in their Granada). On the other hand, it is quite different from study area, which determine the low densities of vector the 15,18% and the 34% detected by MORILLAS-MAR- population and consequent low contact between humans QUEZet al. (1992, 1996) in Almerfa and Axarqufa, res- and vector. pectively. If, as has been pointed out, the methodology In the present study, we have observed that the socio- and serological criteria in their studies are identical to geographic conditions in the Nature Reserve determine a those used in our study, the differences shown can only particular way of life, which probably explains the low be explained according to the generally accepted idea contact between L. infantum and the human population. relating to the distribution of canine leishmaniosis in Thus, we have been able to prove that in the townships microfoci. This observation is confirmed by the results within the Nature Reserve:

Children Adults Total

Acedo-Sanchez et al., 1996. Alpujarra (province of Granada) 32,8% 75,9% 44,1% Morillas-Marquez et aI., 1996. Axarqufa (province of Malaga) 42,4% Alvar et al., 1996. Madrid 1,0% 4,1% 1,4% Sanchfs-Marfn et al., 1997 (province of Almerfa) 1,3% 16,6% 2,4% Present work 0,8% 6,3% 2,9%

Table 5.- Results of leishmanin skin test reported by different authors in the Iberian Peninsula. 118 J.A. LEPE et al. a) it is unusual to find stables in or near houses; Profesor Dr. F. Martine; G6mez (S. Hernandez edit), Universi- b) the majority of parasitised dogs belong to hunting dad de Cordoba: 577-600. GIL-COLLADO (J.), MORILLAS-MARQUEZ (F.) & SANCHlS-MARIN packs, thereby concentrating the focus of the disease; (M.C.), 1989.- Los flebotomos en Espafia. Revista de Sanidad e c) these packs of hunting dogs are kept well away from Higiene Publica, 68: 15-34. townships, thereby avoiding the possibility of esta- HASIBEDER (G.), DYE(C.) & CARPENTIER (1.), 1992.- Matematical blishing the epidemiological cycle dog-sandfly-man, modelling and theory for estimating the basic reproduction which is to say just the opposite of what MORILLAS- number of canine leishmaniasis. Parasitology, 105: 43-53. MARQUEZ et at. (1996) reported from Axarquia (pro- Hoin (R.), 1965.- ouvelles localisations des phlebotornes en Espagne. Annales de Parasitologie Humaine et Comparee, 40: vince of Malaga); 341-363. d) finally, an «empiric» prophylaxis of canine leishma- LUCIE TES-CURDI (J.), SANCHEz-AcEDO (C.), CASTILLO-HERNAN- niosis usually occurs in the Nature Reserve because DEZ (J.A.) & ESTRADA-PENA (A.), 1988.- Sobre la infeccion na- the dogs are generally put down as soon as their hun- tural por Leishmania en Phlebotomus perniciosus Newstead, ting performance declines, which coincides with the 1911, Y Phlebotomus ariasi Tonnoir, 1921, en el foco de leish- first symptoms of leishmaniosis. maniosis de Zaragoza. Revista Iberica de Parasitologia, 48: 7-8. MARTINEZ-CRUZ (M.S.), MARTINEZ (A.), MARTINEZ (F.l.), MARTI- NEZ (F.) & HERNANDEZ (S.), 1990.- Epidemiologfa de la leish- Retrospective study of cases maniosis canina en Cordoba. Revista lberica de Parasitologia, 50: 1-7. The low number of reported cases in humans confirms MARTINEZ-ORTEGA (E.) & CONESA (E.), 1989.- Aproxirnacion al that leishmaniosis in the area is limited to canine foci far funcionamiento epiderniologico de la leishmaniosis en el sureste from inhabited places, which makes the transmission of de la Peninsula Iberica, Revista Iberica de Parasitologia, 49: the disease to humans very difficult. 201-202. MARTINEZ-ORTEGA (E.), ROMERA(H.), CONESA (E.) & GOYENA (M.), 1991.- Comparative study of anthropophilia and phototro- pism among sandflies in a focus of leishmaniosis in the south- ACKNOWLEDGEMENTS eastern Iberian Peninsula. Parassitologia, 33: 413-419. MARTI EZ-ORTEGA (E.), WARD (R.D.), MARTIN(F.) & CONESA Study supported by Research Proyect No. 96/2153 from the (E.), 1982.- Introduccion al estudio de f1ebotomos en Alicante y F.I.S (Fondo de Investigacion Sanitaria), Ministerio de Sanidad y Murcia (Diptera, Phlebotomidae). 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